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First published online 27 May 2003
doi: 10.1242/jcs.00474

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Epidermal tissue regeneration and stromal interaction in HaCaT cells is initiated by TGF-

Division of Differentiation and Carcinogenesis (B0600), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

View larger version (50K): [in a new window]   Fig. 1. Deficient epithelial tissue formation of HaCaT cells in organotypic co-cultures. Epidermal tissue morphology of organotypic co-cultures of normal keratinocytes (NEK) or HaCaT cells grown on collagen type I gels containing 2x105/ml postmitotic fibroblasts throughout a 12-day-culture period (H and E staining; same magnification in all panels; bar, 100 µm).

View larger version (20K): [in a new window]   Fig. 2. (A) Schematic outline of the double paracrine regulation of keratinocyte growth and differentiation by fibroblast interactions (figure modified from Szabowski et al., 2000). (B) Reduced IL-1 expression in HaCaT cultures. Expression levels of IL-1 and IL-1ß in monocultures of postmitotic (irradiated) fibroblasts (HDFi), keratinocytes (NEK) and HaCaT cells as well as in 2D co-cultures of NEK and HaCaT cells, both with HDFi, and in epithelia of organotypic cultures of NEK and HaCaT cells (OTC). Steady-state mRNA levels were determined by semiquantitative RT-PCR using GAPDH expression as internal standard. The RNA expression profile is representative of duplicate determinations of three independent experiments. (C) Lack of IL-1 production in HaCaT cells. Concentrations of IL-1 in supernatants of 3-, 5- and 7-day-old co-cultures with irradiated fibroblasts of NEK and HaCaT cells were determined in aliquots of 2-day-conditioned media by ELISA and calculated as pg/105 cells. Bars represent means ± s.d. of duplicate measurements performed in three independent experiments.

View larger version (35K): [in a new window]   Fig. 3. (A) Deficient production of KGF and GM-CSF in fibroblasts co-cultured with HaCaT cells. Kinetics of KGF and GM-CSF production, measured in culture supernatants of co-cultures with NEK and HaCaT cells, of 2-day-conditioned media, by ELISA and calculated as pg/105 cells. Bars represent means ± s.d. of duplicate measurements performed in three independent experiments. (B) Ineffectiveness of KGF, GM-CSF or IL-1 on epidermal tissue restoration by HaCaT cells in organotypic co-cultures with postmitotic fibroblasts. Organotypic co-cultures of HaCaT cells with postmitotic fibroblasts (3x105/ml) in collagen type-I gel were grown for 10 days in DMEM with 10% FCS (Control) and after addition of IL-1 (5 ng/ml), KGF (10 ng/ml) or GM-CSF (100 ng/ml); medium was changed every second day (H and E staining; bar, 100 µm).

View larger version (21K): [in a new window]   Fig. 4. Decreased cytokine receptor and TGF- expression in HaCaT cells. (A) Expression of KGF and GM-CSF receptors as well as of TGF- were determined in organotypic fibroblastco-cultures of keratinocytes (NEK) and HaCaT cells by semiquantitative RT PCR using GAPDH expression as internal standard. The RNA expression profile is representative of duplicate determinations of three independent experiments. (B) TGF- concentrations in culture supernatants of 3-7-day-old co-cultures of NEK and HaCaT cells with postmitotic fibroblasts were determined in aliquots of 2-day-conditioned media by ELISA and calculated as pg/105cells. Bars represent means ± s.d. of duplicate measurements performed in three independent experiments.

View larger version (66K): [in a new window]   Fig. 5. EGF-receptor signalling is essential for organotypic growth and differentiation of HaCaT cells. (A) Organotypic co-cultures of HaCaT cells with postmitotic fibroblast (3x105/ml) in collagen type-1 gel were grown for 10 days in DMEM as a control (HaCaT), and after addition (every second day) of TGF- (2 ng/ml) or EGF (2 ng/ml) as well as both factors (1 ng each), together with the EGF-receptor blocking antibody C225 (+Ab). For comparison, organotypic co-cultures of normal epidermal keratinocytes were grown for 10 days on collagen gels with 3x105/ml postmitotic fibroblasts (NEK) and with EGF-receptor blocking antibodies (+Ab). (H and E staining; bar, 100 µm). (B) Cytokine and receptor expression in HaCaT epithelia of organotypic co-cultures stimulated for 16 hours with TGF- (2 ng/ml) and EGF, respectively. Expression levels of IL-1, IL-1ß, KGF-R, GM-CSF-R, GM-CSF-Rß, EGF-R and TGF- were determined by semiquantitative RTPCR using GAPDH expression as an internal standard. The RNA expression profile is representative for duplicate determinations of three independent experiments.

View larger version (31K): [in a new window]   Fig. 6. Effects of TGF- on proliferation and apoptosis of HaCaT cells in organotypic co-culture. (A) Proliferation of HaCaT cells in organotypic co-cultures with postmitotic fibroblasts (3x105/ml) grown in DMEM + 10% FCS (control) and after addition of TGF- (2 ng/ml, every second day) was evaluated by counting nuclei stained with an anti-BrdU-specific antibody. Bars represent the percentage of BrdU-positive basal keratinocytes identified and counted on cross-sections. Three vision fields per experiment were counted in three independent experiments. (B) Fragmentation of DNA was detected in organotypic co-cultures grown for 12 days in DMEM with 10% FCS (control) and after addition of TGF- (2 ng/ml, every second day) by TUNEL labeling (red). Nuclei were counterstained with Hoechst DNA dye (blue). In TGF--treated epithelia at day 12, TUNEL-positive cells are localized in the newly formed stratum granulosum and corneum. Bar, 100 µm. (C) Percentage of HaCaT cells with DNA fragmentation in organotypic co-cultures grown for 12 days in DMEM with 10% FCS (control) and after addition of TGF- (2 ng/ml, every second day) was evaluated on cross-sections by counting TUNEL-positive and total number of (bisbenzimide-stained) nuclei. Bars represent percentage of TUNEL-positive cells per epithelium (means ± s.d.). Three vision fields per experiment were counted in three independent experiments. *TUNEL-positive cells at 9 and 12 days are localized in the newly formed stratum granulosum and corneum.

View larger version (80K): [in a new window]   Fig. 7. Normalized tissue architecture of HaCaT organotypic epithelia by TGF-. Morphologic features (H and E staining) of HaCaT organotypic co-cultures with postmitotic fibroblasts (3x105/ml) in collagen type-I gel grown for 10 days in DMEM with 10% FCS (HaCaT) and with TGF- (+ TGF). For comparison, NEK organotypic epithelia were grown and stained under the same conditions. By indirect immunofluorescence microscopy the EGF receptor (EGF-R, red), the early epidermal differentiation marker transglutaminase (TG, red), and the late marker loricrin (Lor, red) have been labeled. Nuclei were counterstained with bisbenzimide (blue). Bar, 100 µm.

View larger version (64K): [in a new window]   Fig. 8. Normalization of epidermal differentiation of HaCaT organotypic co-cultures by cytokines. Epidermal tissue morphology of HaCaT organotypic co-cultures with postmitotic fibroblasts (3x105) in collagen type-1 gel grown for 12 days in DMEM with 10% FCS (control) and treated with 2 ng/ml TGF-. Stimulated cultures were additionally supplemented with KGF (10 ng/ml), GM-CSF (100 ng/ml) or IL-1 (5 ng/ml). For comparison, organotypic co-cultures of normal epidermal keratinocytes (NEK) grown for 12 days on collagen gels with 3x105/ml postmitotic fibroblasts are included (top row: H and E staining). By indirect immunofluorescence the late epidermal differentiation markers filaggrin (red), and loricrin (red) have been labelled. Nuclei were counterstained with bisbenzimide (blue). Bar, 100 µm.